Exon 1 ss of pdgf alpha gene and utilization thereof

ABSTRACT

By using an antisense nucleotide, a ribozyme, a maxizyme, or an RNAi constructed based on the nucleotide sequence of exon 1 beta of the PDGF receptor alpha gene, which is expressed in specific cancer cells, or a polypeptide containing a portion thereof, translation of an mRNA transcribed from exon 1 beta of the PDGF receptor alpha gene is suppressed. An agent for suppressing expression containing as an active ingredient a substance for inhibiting expression, such as an antisense nucleotide, a ribozyme, a maxizyme, or an RNAi, is effective as a therapeutic agent for cancer.

CRROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to Japan PatentApplication No. 2002-332142, filed on Nov. 15, 2002, which isincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to polynucleotides that include exon 1 βof the PDGF receptor α gene or a part thereof, to methods, substances,and agents for suppressing expression of PDGF receptor α which targetmRNA including exon 1 β among mRNAs of the PDGF receptor α gene and tocancer therapeutic agents.

BACKGROUND ART

Platelet-derived growth factor (PDGF) plays an important role in cellproliferation, development and differentiation, wound healing, malignantprogression of cancer and arteriosclerosis, etc. It is thereforeexpected that regulation of PDGF signaling will lead to discovery oftherapeutic agents for these diseases. As therapeutic agents, in fact,PDGF expression suppressors (refer to, e.g., Japanese Laid-OpenApplication No. 10-59850), inhibitors of binding between PDGF and PDGFreceptor α (National Publication of International Patent Application No.1996-13370), and tyrosine kinase inhibitor of PDGF receptor α (refer to,e.g., National Publication of International Patent Application No.2002-514228) have been proposed.

However, these suppressors and inhibitors can affect not onlycancer-specific PDGF signaling but also normal PDGF signaling. Thus, theobject of the present invention is to provide polynucleotides,substances for suppressing expression, agents for suppressingexpression, and cancer therapeutic agents for use in methods forsuppressing expression that enable selective suppression of PDGF signalsspecific to cancer cells.

DISCLOSURE OF THE INVENTION

The polynucleotide according to the present invention has the nucleotidesequence shown in SEQ ID NO: 2 or a part thereof. An example of apolynucleotide having a part of the nucleotide sequence shown in SEQ IDNO: 2 is, for example, the one having the nucleotide sequence shown inSEQ ID NO: 1.

Further, the polynucleotide according to the present invention has thenucleotide sequence of SEQ ID NO: 2 with one or a few nucleotidesdeleted, substituted, or added, or a part thereof, which is included inthe nucleotide sequence of the sense strand of the PDGF receptor α gene.

“PDGF receptor α gene” as used herein refers to the gene composed of thenucleotide sequence shown in GenBank Accession Nos. AC026580 andAC025013 and its homologues.

Further, the polynucleotide according to the present invention may be apolynucleotide that has a nucleotide sequence complementary to theaforementioned polynucleotide or part thereof.

These polynucleotides may be any one of double-stranded DNA,single-stranded DNA, double-stranded RNA, and single-stranded RNA.Further, a polypeptide that has a genetic polymorphism which isdeletion, substitution, or addition of one or a few nucleotides, andthat has a nucleotide sequence included in the nucleotide sequence ofthe sense strand of the PDGF receptor α gene or a part thereof is withinthe scope of the present invention. However, the polypeptide ispreferred to have an equivalent function and is more preferred to betranscriptionally regulated by E2F-1.

The method for suppressing expression of PDGF receptor α according tothe present invention targets mRNA containing exon 1 β among mRNAs ofthe PDGF receptor α gene. “mRNA” as used herein refers to RNA formed byremoving the introns from hnRNA and linking the exons. “To target anmRNA” as used herein refers to specifically preventing, directly orindirectly from the mRNA, formation of its encoded protein. “Proteinexpression” refers to production of proteins as a result of accuratetranslation of genetic information on DNA through mRNA.

The method for suppressing expression of PDGF receptor α according tothe present invention may be any one that uses an antisense nucleotide,a ribozyme, a maxizyme, or an RNAi.

“Antisense nucleotide of the PDGF receptor α gene” as used herein refersto a nucleotide complementary to the nucleotide sequence of mRNA of thePDGF receptor α gene. The antisense nucleotide of the PDGF receptor αgene may be an antisense RNA or an antisense DNA, and modifiednucleotides may be used. The above-mentioned antisense RNA or DNA refersan RNA or a DNA complementary to a mRNA sequence transcribed from atarget gene. An antisense RNA or DNA is used to block expression ofgenetic information in a cell and to specifically suppress production ofthe target protein.

“Ribozyme” is the general term of RNA with enzyme activity; it refers toan enzyme that specifically cleaves organism-based RNA. A ribozyme hasthe function of cleaving a target RNA sequence when taken up into cells,resulting in suppression of protein expression from the target RNA. Aribozyme is preferable as a substance for suppressing protein expressionbecause of its high specificity to a target RNA sequence. Ribozymesinclude a hammerhead ribozyme, hairpin ribozyme, etc.

“Maxizyme” is generally RNA molecules that form the dimer structure asdescribed in WO99/46388. For example, it is possible to cleave onlycancer cell-specific mRNAs by constructing a maxizyme so that its twoRNA molecules recognize cancer cell-specific mRNAs, instead ofrecognizing mRNA in a normal cell.

“RNAi” is a technique using double-stranded RNA (dsRNA) that induces aphenomenon called RNA interference. “Phenomenon called RNA interference”refers to a phenomenon in which expression of a target gene issuppressed when the double-stranded RNA is introduced into a cell.Currently, RNAi is considered to work like this: an endogenous mechanismin a host cuts an RNA molecule into 21-23 base-pair short RNAs. Theseshort RNA molecules recognize and sequence-specifically degrade mRNAtranscribed from a host gene. As a result, expression of the proteinencoded by the host gene is specifically suppressed.

The method for suppressing expression according to the present inventionmay be any one that uses DNA that encodes an antisense RNA, a ribozyme,a maxizyme, or an RNAi.

The substance for suppressing expression of PDGF receptor α according tothe present invention targets a mRNA containing exon 1 β among mRNAs ofthe PDGF receptor α gene. This is caused, for example, by binding of asubstance for suppressing expression to the mRNA or its degradation ofthe mRNA. Furthermore, a substance for suppressing expression mayindirectly cause some other substance to bind to the mRNA or to degradethe mRNA. It should be noted that “substance for suppressing expressionof PDGF” refers to a substance that suppresses production of the PDGFreceptor α protein from the PDGF receptor α gene.

Specific examples of the substance for suppressing expression of PDGFreceptor α according to the present invention include an antisensenucleotide, a ribozyme, a maxizyme, or an RNAi.

Specific examples of the substance for suppressing expression of PDGFreceptor α according to the present invention include DNA that encodesantisense RNA, a ribozyme, a maxizyme, or an RNAi.

The agent for suppressing expression of PDGF receptor a according to thepresent invention contains the aforementioned substance for suppressingexpression as an active ingredient.

The therapeutic agent for cancer according to the present inventioncontains the aforementioned agent for suppressing expression.

The therapeutic method for cancer according to the present inventionuses the aforementioned agent for suppressing expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression patterns of exons 1 β to 4 of the PDGFreceptor α gene, examined using RT-PCR in Example 2 of the presentinvention.

FIG. 2 shows the structure in a ribozyme.

FIG. 3 shows the structure in a maxizyme.

FIG. 4 shows a result of luciferase assay using a reporter constructincluding the sequence from nucleotides −1395 to +312 in Example 1according to the present invention. “+” in the figure shows a result ofintroduction of 100 ng of the reporter construct including the sequencefrom nucleotides 1395 to +312. “−” in the figure shows a result ofintroduction of 100 ng of the reporter construct lacking the sequencefrom nucleotides −1395 to +312.

FIG. 5 shows a result of luciferase assay using a reporter constructincluding the sequence from nucleotides −517 to +1445 in Example 1according to the present invention.

FIG. 6 shows a result of luciferase assay using deletion mutants ofvarious lengths that lack their transcription start points in Example 1of the present invention.

FIG. 7 schematically shows mRNA of PDGFRα transcribed by basictranscription factors and mRNA of PDGFRα transcribed by E2F-1.

BEST MODE FOR CARRYING OUT THE INVENTION

It is known that in cancer cells, in most cases, abnormalities haveoccurred in the signal transduction pathway in which cancer suppressorproteins, such as the RB protein, are involved. For example,overexpression of cycline D1, which functions upstream of the RBprotein, is considered to contribute to malignant progression of cancercells by enhancing sensitivity to growth factors. In an in vitro cellculture system, by adding fibroblast growth factor (FGF) to a cell linein which cycline D1 is overexpressed, the cells become malignant. Theinventors found out that platelet-derived growth factor (PDGF) alsofunctions in the same manner—i.e., cause a cell line in which cycline D1is overexpressed to become malignant.

The transcription factor E2F-1 is present downstream in the cyclineD1-RB pathway and enhances its sensitivity to FGF by enhancingexpression of FGF receptors. E2F-1 enhances expression of PDGF receptorα as well but the inventors found that E2F-1 does not act on the knownpromoter. The inventors found out a novel promoter region regulated byE2F-1 in the conventional intron 1 and identified novel exon 1 β to beused in transcription by the novel promoter, as will be described indetail in Examples.

Thus, it has been shown that, in the malignant progression of cancerinvolving E2F-1 and PDGF, one of the targets of these factors is mRNAthat contains exon 1 β of PDGF receptor α. Consequently, by specificallyinhibiting production of PDGF receptor α from transcripts having thisexon 1 β, it is possible to block the signaling pathway leading tomalignant progression of cancer cells and thereby to suppressproliferation of cancer cells. It should be noted that, whenproliferation of cancer cells is induced by some factor that cause thecancer to be malignant in collaboration with PDGF receptor α by inducingovertranscription of mRNA containing exon 1 β, the present invention isapplicable to inhibition of proliferation of such cancer cells, even ifthe cycline D1-E2F-1 pathway is not involved.

Embodiments of the present invention accomplished based on theabove-described findings are hereinafter described in detail by givingExamples. Unless otherwise explained, methods described in standard setsof protocols such as J. Sambrook and E. F. Fritsch & T. Maniatis (Ed.),“Molecular Cloning, a Laboratory Manual (2nd edition), Cold SpringHarborPress and Cold Spring Harbor, N.Y. (1989); and F. M. Ausubel, R.Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K.Struhl (Ed.), “Current Protocols in Molecular Biology,” John Wiley &Sons Ltd., or alternatively, their modified/changed methods are used.When using commercial reagent kits and measuring apparatus, unlessotherwise explained, protocols attached to them are used.

The object, characteristics, and advantages of the present invention aswell as the idea thereof will be apparent to those skilled in the artfrom the descriptions given herein. It is to be understood that theembodiments and specific examples of the invention described hereinbelow are to be taken as preferred examples of the present invention.These descriptions are only for illustrative and explanatory purposesand are not intended to limit the invention to these embodiments orexamples. It is further apparent to those skilled in the art thatvarious changes and modifications may be made based on the descriptionsgiven herein within the intent and scope of the present inventiondisclosed herein.

A polynucleotide having the nucleotide sequence of the exon 1 β of thehuman PDGF receptor α gene shown in SEQ ID NO: 2 or a part thereof canbe prepared, based on nucleotide sequence information shown in SEQ IDNO: 2, from human a gene library, such as a cDNA library or a genomiclibrary. In addition, exon 1 β of the PDGF receptor α gene derived fromorganisms other than humans, such as mice, rats, chicks, pigs, dogs, andmonkeys can be also defined by being transcriptionally regulated byE2F-1. It can also be identified, for example, by hybridization to exon1 β of the human PDGF receptor α gene or examination of a new exon inthe conventionally known intron 1 sequence. The exon 1 β of the PDGFreceptor α gene derived from organisms other than humans is also amongthe polynucleotides according to the present invention.

“Exon 1 β of the human PDGF receptor α gene” as used herein refers tothe exon including the polynucleotide that has the nucleotide sequenceshown in SEQ ID NO: 2. “Exon 1 β of the PDGF receptor α gene” refers tothe exon containing the polynucleotide that has the nucleotide sequenceshown in SEQ ID NO: 2 and the exon corresponding thereto in speciesother than humans.

Being the non-coding region on mRNA, exon 1 β of the PDGF receptor αgene does not necessarily need to have a high homology at the nucleotidesequence level in species other than humans, but it needs to conservethe functional aspect of being transcribed in a specific cell type; forexample, it needs to have the feature that its transcription isregulated by E2F-1, which another exon 1 does not have.

As shown in FIG. 1, mRNA containing exon 1 β among mRNAs of the PDGFreceptor α gene is detected only in specific cancer cells. Therefore,the method for suppressing expression that targets exon 1 β-containingmRNA among mRNAs of the PDGF receptor α gene, the polynucleotide, thesubstance for suppressing expression, the agent for suppressingexpression, etc. according to the present invention are useful astherapeutic methods and agents for cancer for the purpose of attackingspecific cancer cells (illustratively, e.g., human colon cancer cellsSW480, human esophageal cancer cells T.Tn, etc.)

The method for suppressing expression according to the present inventiontargets mRNA containing exon 1 β among mRNAs of the PDGF receptor αgene. It may be, for example, the method for suppressing expression ofPDGF receptor α by causing the mRNA containing exon 1 β among mRNAs of aPDGF receptor α gene to be bound, thereby suppressing translation.Alternatively, it may be the method for suppressing expression of PDGFreceptor α by degrading or cleaving the above-mentioned mRNA. In thefollowing, the methods for suppressing expression using substances forsuppressing expressions, such as an antisense nucleotides, a ribozyme, amaxizyme, or an RNAi, will be described by means of examples.

(1) Preparation of Antisense Nucleotides

The antisense nucleotides for use in the method for suppressingexpression according to the present invention are illustrativelyantisense RNA or antisense DNA having the nucleotide sequencecomplementary to the nucleotide sequence of the portion corresponding toexon 1 β among mRNAs of the PDGF the nucleotide sequence complementaryto part thereof. In this case, an antisense oligonucleotide consistingof a 15-30 nucleotide sequence may be used.

The antisense nucleotide according to the present invention is notlimited, in terms of structure, to the location or length of itssequence, modifications, or presence of mismatches in the sequence. Theabove-mentioned modified antisense nucleotides are illustratively theantisense nucleotide linked by phosphodiester bonds that have aphosphate group with one oxygen atom modified with a sulfur atom or amethyl group for stabilization of antisense nucleotides in the body orcells or the morpholino-modified antisense nucleotide.

The antisense DNA according to the present invention can be synthesizedin vitro by the primer extension method using a DNA-dependent DNApolymerase such as S1 nuclease. It is also possible to synthesize onlyantisense strands by performing PCR using a part of the antisense strandas primers. Alternatively, antisense DNA may be synthesized artificiallyby using a DNA synthesizer or other means. Antisense oligonucleotidesare synthesized in the same manner as antisense DNA and are mostpreferably artificially synthesized.

On the other hand, the antisense RNA according to the present inventioncan easily be synthesized with an RNA synthesizer, by solid-phasesynthesis, or other means. Subsequently, target RNA can be isolated byelution with NH3OH/EtOH using HPLC.

The antisense RNA according to the present invention may be artificiallysynthesized as described above, but it may be synthesized in an in vitrotranscription system with T7 RNA polymerase by constructing a vectorinto which a double-stranded DNA having a DNA sequence corresponding toantisense RNA has been inserted downstream of the promoter sequence(5′-TAATACGACTCACTATA-3′: SEQ ID NO: 3) specifically recognized by T7RNA polymerase.

Alternatively, antisense RNA may be expressed in cells using expressionvectors, such as a virus vector or a plasmid incorporating DNAcorresponding to the antisense RNA. The virus vector may be anadenovirus vector or a retroviral vector.

(2) Preparation of a Plasmid into which a Ribozyme has Been Inserted

A ribozyme has nucleotide sequences (sequences at the 5′ and 3′ ends ofa ribozyme are shown) complementary to the target mRNA sequences and a24-nucleotide sequence including the catalytic active site (circlednucleotides), as shown in FIG. 2. When the 5′ and 3′ end sequences of aribozyme specifically bind to the target mRNA sequence, it is possibleto cleave mRNA at sequence NUX (where N can be any nucleotide and X canbe A, U or C) and thereby to degrade the target mRNA. Accordingly, it isconsidered that by synthesizing a ribozyme using as a substrate thenucleotide sequence of the portion corresponding to exon 1 β among mRNAsof the PDGF receptor a gene and administering such a ribozyme intocells, it is possible to specifically cleave mRNAs containing Exon 1 βamong mRNAs of the PDGF receptor α gene.

Therefore, the ribozyme that is the substance for suppressing expressionaccording to the present invention can be designed such that, afterselecting the sequence corresponding to an NUX sequence from thenucleotide sequence of exon 1 β of the human PDGF receptor α gene shownin SEQ ID NO: 2, both a nucleotide sequence complementary to a 15-20nucleotide sequence containing the selected sequence and a 24-nucleotidesequence including the catalytic active site (circled nucleotides) areincluded in the ribozyme. Specific examples of the sequencecorresponding to an NUX sequence include, for example, GTCs at positions34 to 36, GTCs at positions 75 to 77, GTCs at positions 78 to 80, GTCsat positions 81 to 83, GTCs at positions 172 to 174, and GTCs atpositions 267 to 269 shown in SEQ ID NO: 2. The above-mentioned ribozymemay be a hammerhead ribozyme and a hairpin ribozyme.

The actual methods for synthesizing the ribozyme include artificialsynthesis, in vitro synthesis, intracellular synthesis with anexpression vector, etc., and as the techniques are the same as thoseused for antisense RNA synthesis as described in (1), their explanationis omitted here.

(3) Design and Preparation of a Maxizyme

As shown in FIG. 3, a maxizyme is composed of RNA molecules that formthe dimer structure. Each of the two RNA molecules has a sensor arm (X .. . X region in the figure, X representing any nucleotide. X in theupper row and the corresponding X in the lower row represent a pair ofcomplementary nucleotides.) that specifically recognizes a target RNA; acatalytic active site (the region where double strands are not formed inthe figure); and a site that recognizes an NUX sequence (where N can beany nucleotide and X can be A, U or C; represented as the sequence GUCin the figure) on the mRNA containing the target RNA and upstream(upstream of the sequence GUC in the figure) or downstream (downstreamof the sequence GUC portion in the figure) thereof. When a sensor arm ofa maxizyme has specifically recognized and bound to the target RNA, andanother sensor arm of the maxizyme has specifically recognized and boundto the upstream and downstream of an NUX sequence on the mRNA containingthe target RNA, the maxizyme can cleave the NUX sequence on the mRNAcontaining the target RNA, thereby degrading the target mRNA.Accordingly, it is considered that by synthesizing a maxizyme on thebase of the nucleotide sequence of the portion corresponding to exon 1 βin mRNA of the PDGF receptor α gene and administering such a maxizymeinto cells, it should be possible to specifically cleave mRNA containingexon 1 β among mRNAs of the PDGF receptor α gene.

For example, exon 1 β of the PDGF receptor α gene is used as the targetRNA and a nucleotide sequence complementary to this target RNA as asensor arm. An NUX sequence that is present downstream of the target RNAis then selected from mRNA of PDGF receptor α and the nucleotidesequence complementary to the sequences upstream and downstream of theNUX sequence is determined. The NUX sequence may be the one on mRNA ofexon 1 β of the PDGF receptor α gene. Based on these pieces ofinformation, a maxizyme can be prepared by synthesizing each RNAmolecule of the intended maxizyme with an RNA synthesizer. There may beone or a few increases or decrease in the number of the Xs shown in thefigure.

The actual methods for synthesizing the ribozyme include artificialsynthesis, in vitro synthesis, intracellular synthesis with anexpression vector, etc. and as the techniques are the same as those usedfor antisense RNA synthesis as described in (1), their explanation isomitted here. It should be noted that, since a maxizyme uses two RNAmolecules, when a maxizyme is expressed in cells by means of expressionvectors, two RNA molecules may be incorporated into one vector or eachRNA molecule may be separately incorporated into two vectors.

(4) Preparation of RNAi

It was reported that introduction of double-stranded RNA correspondingto a gene of interest into an organism causes degradation of thecorresponding mRNA (Bass, B. L. (2000) Cell 101, 235-238, Fire, A.(1999) Trends Genet. 15, 358-363, Sharp, P. A. (2001) Genes Dev. 15485-490). It is therefore considered that, when double-stranded RNA(RNAi) corresponding to the mRNA containing exon 1 β of the PDGFreceptor α gene or a part thereof is introduced into cells or anorganism, mRNA containing exon 1 β among mRNAs of the PDGF receptor αgene is degraded.

RNAi that is a substance for suppressing expression according to thepresent invention can be prepared as follows. RNA having the nucleotidesequence of the sense strand of exon 1 β of the PDGF receptor α gene ora part thereof and RNA complementary to the RNA can be artificiallysynthesized using an RNA synthesizer, or may be synthesized in vitro andin vivo using HiScribeRNAi Transcription Kit (manufactured by NEB).

Alternatively, by introducing, into cells, expression vectors, such asvirus vectors or plasmids, into each of which exon 1 β of the PDGFreceptor α gene or a part thereof has been cloned in positive ornegative direction, and expressing both strands of DNA in cells, RNAi isformed in the cells and the target mRNA is degraded. An expressionvector can be used including DNA having a sequence which has resultedfrom fusion of the DNA sequences of each strand of the double strandcorresponding to exon 1 β of the PDGF receptor α gene or a part thereof,i.e., DNA having a sequence in which the 3′ end of the sense strand DNAhas been fused to the 5′ end of the antisense strand DNA or DNA having asequence in which the 3′ end of the complementary DNA has been fused tothe 5′ end of the sense strand DNA. The above-mentioned virus vector maybe an adenovirus vector, a retroviral vector, or the like. These RNAisare preferably of up to 30 bases long, most preferably, of up to 21bases long.

(5) Introduction of a Substance for Suppressing Expression

In a in vitro cell culture system, for intracellular introduction of asubstance for suppressing expression, a prepared substance forsuppressing expressions, such as an antisense nucleotide, a ribozyme, amaxizyme, or an RNAi, is introduced into the intended cancer cells bythe electroporation method, microinjection method, lipofection method,viral infection method using a viral vector (e.g., an adenovirus or aretrovirus), transfection method using calcium, or the like.

On the other hand, as the method for introducing an agent forsuppressing expression to an individual in vivo, an agent forsuppressing expression that contains as an active ingredient a substancefor suppressing expressions, such as the aforementioned preparedantisense nucleotide, ribozyme, maxizyme, or RNAi may be directlyadministered to the vicinity of targeted cancer cells in a human or avertebrate other than a human. Alternatively, depending on the agent,parenteral, oral, intradermal, subcutaneous, intravenous, intramuscular,or intraperitoneal administration may be performed. In this case, anagent for suppressing expression may further contain a suitablepharmacologically acceptable excipient or base, depending on the site orpurpose of administration.

An agent for suppressing expression to be administered to an individualis preferably prepared such that the substance for suppressingexpression is easily taken up into cells. As one possible method, forexample, an expression vector made by integrating the aforementionedantisense nucleotide, ribozyme, maxizyme, or RNAi into a viral vector isinfected into a suitable cell line invitro so that it produces thevirus, and the produced virus can be used for infection by injection.The virus vector to be used may be an adenovirus vector or a retroviralvector which can function in cells.

In addition, a plasmid may be introduced into cells by encapsulating theaforementioned expression vector in a liposome so that it fuses tocancer cells. Alternatively, in vivo transfection may be performed usingTransIT In Vivo Gene Delivery System (TAKARA). In this case, the agentfor suppressing expression may be directly injected into the affectedsite or intravenously injected.

Alternatively, an RNA aptamer in which the aforementioned antisensenucleotide, ribozyme, a maxizyme, or RNA such as RNAi has been bound topeptides, such as HIV TAT, which are easily introduced into cells, maybe injected as an agent for suppressing expression by an in vitroselection method. Cancer cells to be targeted are illustratively humanSW 480 colon cancer cells or human T.Tn esophageal cancer cells, butthey are not limited to any specific ones, as long as they are cancercells in which mRNA transcribed from exon 1 of the PDGF receptor α genecan be detected.

(6) Asessment of Suppression of Expression by a Substance forSuppressing Expression

According to the methods described in the previous (5), by preparingspecific cancer cells cultured in vitro with or without a substance forsuppressing expression and by comparing and evaluating the amount ofmRNA transcribed from exon 1 β of the PDGF receptor α gene by the RT-PCRmethod, suppression of expression by a substance for suppressingexpression can be assessed. Alternatively, a method for assessing theamount of mRNA transcribed from exon 1 β of the PDGF receptor α gene bynorthern blotting etc. may be used. Based on these results, antisensenucleotides capable of suppressing more effectively translation of mRNAtranscribed from exon 1 β of the PDGF receptor α gene can be found.

Although the above-mentioned procedure is for in vitro experiment, it ispossible to make an assessment in in vivo experiment as well.

The in vivo assessment method is illustratively the following: Theaforementioned specific cancer cells are subcutaneously injected intonormal mice, the tumor is allowed to grow during a particular period oftime, and subsequently, the aforementioned agent for suppressingexpression prepared by the method described (5) is injected as single ormultiple doses. Following injection(s), suppression of expression by thesubstance for suppressing expression can be assessed by comparing andevaluating tumor sizes and survival rates between the treated mice andnon-treated mice.

Examples according to the present invention will be described in detailhereinbelow.

EXAMPLE 1

In this example, the novel exon regulated by the transcription factorE2F-1 in the PDGF receptor α gene was identified.

First, the inventor found out that mouse NIH 3T3 cells proliferate byaddition of PDGF, in a manner not depending on the scaffold. Thus,expression of PDGF receptor α (PDGFR-α) in the mouse NIH 3T3 cell linewas examined and it was found that the expression of PDGF receptor α wasenhanced, being regulated at the transcriptional level.

Since cycline D1 activates the transcription factor E2F-1 through cellcycle-dependent pRb phosphorylation, it was investigated whether or notthis enhancement was due to the regulation of the promoter of human PDGFreceptor α by E2F-1. The promoter region consisting of sequences from−1395 to +312 (nucleotides numbered according to the transcription startpoint reported in Genomics, Vol. 30, 224-232, 1995. Refer to GenBankAccession No. D50001S01) relative to the transcription start point wascloned upstream of a luciferase gene using the pGL3 luciferase vectorand introduced into cycline D1-overexpressed NIH 3T3 cells together withan E2F-1 expression vector by transfection. After culture for 24 to 72hours, luciferase assay was performed to examine transcriptionalactivity by the above-mentioned promoter. As a positive control, aplasmid containing a luciferase gene downstream of the mFGFR-1 promoterwas used. As a result, transcriptional activity of the mFGFR-1 promoterwas enhanced, whereas transcriptional activity of the PDGF receptor αpromoter was not enhanced (FIG. 4). These results revealed that theconventionally known PDGF receptor α promoter is not involved inenhancement of expression of the PDGF receptor α in mouse cyclineD1-overexpressed NIH 3T3 cells.

Then, it was examined whether or not a consensus sequence that binds toE2F-1 is present downstream of exon 1 of the human PDGFR-α gene by thesearch for transcription factor binding sequences (TF search). It wasfound that sequences to which E2F-1 would bind are present at fourlocations in clusters near approximately 1 kbp downstream of thereported transcription start point. Thus, a DNA consisting of thesequence from nucleotides −295 to +1445 was inserted upstream of aluciferase gene. By using the obtained reporter construct, luciferaseassay was performed in the same manner previously described to examinetranscriptional activity by E2F-1. The result confirmed that thetranscriptional activity in this region is enhanced by E2F-1 (FIG. 5).Further, no transcription activity was detected with an E2F-1 mutant(amino acid sequence from 1 to 368) defective in E2F-1 transcriptionactivation or another E2F-1 mutant defective in DNA binding abilityconstructed by substituting leucine 132 for glutamic acid, suggestingthat the region is in fact regulated by E2F-1.

However, since the putative E2F-1 binding site is present about 1.2 kbpdownstream of the transcription start point previously reported, thispromoter activity is unlikely to act on the transcription start pointpreviously reported. Hence, to examine whether the region is working onthe transcription start point previously reported, deletion mutants ofvarious lengths lacking this transcription start point were constructedand luciferase assay was performed in the same manner as above describedto examine transcriptional activity by E2 F-1. As a result, even theconstruct deleted to about 1 kbp downstream of the transcription startpoint had promoter activity and further exhibited enhancement oftranscriptional activity by E2F-1 as well. These results suggested apossibility that mRNA regulated by E2F-1 of PDGFR-α might be expressedat a new transcription start point (FIG. 6).

Then, the transcription start point associated with E2F-1 was determinedby the 5′RACE method. mRNA was extracted from the NIH 3T3 cell line intowhich the reporter construct in which DNA having the sequences fromnucleotides −295 to +1445 was inserted upstream of a luciferase gene hadbeen introduced by transfection. PCR was performed with primers (forwardprimer 1 [SEQ ID NO: 4:5′-CCT TAATTAAGGGATTCTCGCATGCCAGAGATCCTA-3′];reverse primer 1 [SEQ ID NO: 5:5′-CCTTAATTAAGGGGCGCAACTGCAACTCCGATAAAT-3′]) specific to the luciferase gene sequence by using 5′Full RACECore Set (TAKARA's brand name), yielding amplified products. Examinationof the DNA sequences of these amplified products revealed the presenceof a novel exon in a region previously regarded as an intron. Thus, itwas shown that a novel exon transcriptionally regulated by E2F-1 ispresent in a region previously considered an intron.

EXAMPLE 2

In this Example, it was examined if exon 1 β of the PDGF receptor α geneis specifically expressed in cancer cells.

Primers (forward primer 2 [SEQ ID NO:6:5′-CCTTAATTAAGGAACCGCACACCAAGGGGCCCTCATT-3′); reverse primer 2 [SEQ IDNO: 7:5′-AACAGCACAGGTGACCACAATCG-3′]) were designed from the sequencesof exon 1 β of the PDGF receptor α gene and the previously known exon 4.The RT-PCR was performed using the total RNAs extracted from variouscancer cells shown in FIG. 1. As shown in FIG. 1, signals were detectedin SW480 human colon cancer cells and human T. Tn oesophageal cancercells. These amplified bands were recovered and the DNA sequences wereexamined. The results revealed that these bands were mRNA fragments ofhuman PDGFR-α in which the sequences of exon 1 β of the PDGF receptor αgene and the previously known exons 2 to 4 are linked together.Simultaneously, the 338 bp full-length sequence of exon 1 β shown in SEQID NO: 1 was determined. Namely, the newly identified promoter regionwas revealed to belong to Human PDGFR mRNA2 (FIG. 7).

EXAMPLE 3

More exact location of 5′ end of exon 1 β was examined by 5′RACE method.mRNA was extracted from human MG-63 osteosarcoma cells expressingPDGFR-α mRNA including exon 1 β. By using 5′Full RACE Core Set, PCR wasperformed with primers (forward primer 2 [SEQ ID NO: 6]; reverse primer3 [SEQ ID NO: 8: 5′-CCGCTCGAGGCGACGACGACTTCTTCACTCAGG-3′] specific toexon 1 β. DNA sequencing of amplified products obtained by this PCRrevealed that the 363 bp nucleotide sequence shown in SEQ ID NO: 2 hadbeen obtained and 5′ end of exon 1 β extended to at least +1210.

INDUSTRIAL APPLICABILITY

As described above, according to the present invention, polynucleotides,substances for suppressing expression, agents for suppressingexpression, and cancer therapeutic agents, for use in methods forsuppressing expression that enable selective suppression of PDGF signalsspecific to cancer cells, can be provided.

1. A polynucleotide comprising the nucleotide sequence shown in SEQ IDNO: 2 or a part thereof.
 2. A polynucleotide comprising a nucleotidesequence shown in SEQ ID NO: 2, in which one or more nucleotides aredeleted, substituted, or added, comprising a nucleotide sequencecontained in the nucleotide sequence of the sense strand of the PDGFreceptor alpha gene or a part thereof.
 3. A polynucleotide comprising anucleotide sequence complementary to the polynucleotide or part thereofof claim
 1. 4. A method for suppressing expression of PDGF receptoralpha comprising targeting mRNA including exon 1 beta among mRNAs of thePDGF receptor alpha gene.
 5. The method of claim 4, wherein antisensenucleotides, a ribozyme, a maxizyme, or an RNAi is used.
 6. The methodof claim 4, wherein DNA that encodes an antisense RNA, a ribozyme, amaxizyme, or an RNAi is used.
 7. A substance for suppressing expressionof PDGF receptor alpha comprising targeting mRNA containing exon 1 betaamong mRNAs of the PDGF receptor alpha gene.
 8. The substance of claim7, which is antisense nucleotides, a ribozyme, a maxizyme, or an RNAi.9. The substance of claim 7, which is a DNA that encodes an antisenseRNA, a ribozyme, a maxizyme, or an RNAi.
 10. An agent for suppressingexpression of PDGF receptor alpha comprising the substance of claim 7 asan active ingredient.
 11. A therapeutic agent for cancer comprising theagent of claim
 10. 12. A therapeutic method for cancer, wherein theagent of claim 10 is used.
 13. A polynucleotide comprising a nucleotidesequence complementary to the polynucleotide or part thereof of claim 2.